The present invention relates generally to methods for controlling expression of a foreign gene. In particular, the present invention relates to methods for inducing sustained gene expression by a single application of stress.
Exposure of cells, tissues and organs to xe2x80x9cstress,xe2x80x9d such as elevated temperature, heavy metals, oxidants, chemicals interfering with mitochondrial function, alcohols, hypoxia, hyperosmotic and hypoosmotic environments, amino acid analogues, and benzoquinone ansamycins results in the activation or enhanced activity of a group of genes known as heat shock or stress (hsp) genes. See, for example, Scharf et al., xe2x80x9cHeat Stress Promoters and Transcription Factors,xe2x80x9d in Results and Problems in Cell Differentiation 20, Nover (Ed.), pages 125-162 (Springer-Verlag 1994). When cells, tissues and organs are returned to a normal condition, stress gene activity declines, until it reaches the low pre-stress level.
Stress genes encode a small number of heat shock or stress protein (Hsp) families. Major families of stress proteins are distinguished on the basis of molecular weight and amino acid sequence. See, for example, Nover and Scharf, Cell. Mol. Life Sci. 53:80 (1997). They include Hsp110 (Hsp""s with a subunit molecular weight of about 110 kDa), Hsp104, Hsp90, Hsp70, Hsp60, Hsp27, Hsp10 and ubiquitin. Many of these Hsp""s are molecular chaperones that participate in such basic cellular processes as protein folding, protein degradation and protein trafficking.
Promoter regions of stress genes typically include so-called heat shock element (HSE) sequences. These sequences are essential to render the genes activatable by stress. HSE provide binding sites of proteins named heat shock transcription factors (HSF). See, for example, Wu, xe2x80x9cHeat Shock Transcription Factors: Structure and Regulation,xe2x80x9d in Annu. Rev. Cell Dev. Biol. 11:441 (1995); Nover and Scharf, Cell. Mol. Life Sci. 53:80 (1997). Mammalian cells can express at least three different HSF. It is thought that the factor termed HSF1 is responsible for the regulation of hsp genes by stress. HSF1 is continuously and ubiquitously expressed in mammalian cells. In the absence of stress, the factor is present in an inactive form, unable to bind HSE sequences of stress gene promoters and to enhance their transcription. During stress, HSF1 is activated, and in the activated form, it binds HSE DNA and stimulates transcription of stress genes. Subsequent to a stressful event, the factor relatively rapidly returns to its inactive form. Consequently, transcription of stress genes ceases. Mutant forms of HSF1 have been constructed that are capable of constitutively transactivating stress genes, in the absence of stress.
Promoters of stress genes have been linked to genes of interest to render the genes activatable by stress. Constructs of this kind were used to prepare cell lines, in which the gene of interest could be activated by heat or some other form of stress. For example, cell lines have been prepared that contain a stress gene promoter-controlled growth hormone gene. Dreano et al., Gene 49:1 (1986). Moreover, transgenic flies and transgenic nematodes have been produced that express a xcex2-galactosidase gene under stress control. See, for example, Voellmy and Ananthan, U.S. Pat. No. 5,346,812, and Candido and Jones, Trends Biotechnol. 14:125 (1996), and Jones et al., Toxicology 109:119 (1996).
A major drawback of the use of stress promoters to control regulation of a gene of interest is that gene expression induced by a stress promoter can be maintained beyond the duration of the stress treatment only under conditions of extreme stress. Yet such extreme conditions are incompatible with cell survival.
Therefore, a need exists for a means to take advantage of stress-induced gene regulation under conditions that do not endanger cell viability.
The present invention provides molecular circuits that can be activated by stress, and that regulate expression of a gene of interest.
In particular, the present invention provides molecular circuits comprising (a) a first nucleic acid molecule that comprises a gene encoding a transcription factor and a first promoter activatable by stress and by the transcription factor, wherein the first promoter and the transcription factor gene are operably linked, and (b) a second nucleic acid molecule that comprises a gene of interest and a second promoter activatable by the transcription factor, wherein the second promoter and the gene of interest are operably linked. In a variation of this type of molecular circuit, the molecular circuit comprises a gene of interest that encodes a transactivator, and the molecular circuit further comprises a nucleic acid molecule comprising a second gene of interest and a promoter activatable by the transactivator, wherein the second gene of interest and the transactivator-activatable promoter are operably linked. The molecular circuits may comprise two separate nucleic molecules, or the molecular circuits may comprise a single nucleic acid molecule that contains the first and second nucleic acid molecules.
The present invention also includes molecular circuits comprising (a) a first nucleic acid molecule that comprises a gene encoding a transcription factor and a first promoter activatable by stress, wherein the first promoter and the transcription factor gene are operably linked, (b) a second nucleic acid comprising a gene encoding the transcription factor and a second promoter activatable by the transcription factor, wherein the second promoter and the transcription factor gene are operably linked, and (c) a third nucleic acid molecule that comprises a gene of interest and a third promoter activatable by the transcription factor, wherein the third promoter and the gene of interest are operably linked. These molecular circuits may comprise (a) three separate nucleic acid molecules, (b) the third nucleic acid molecule and a single nucleic acid molecule that comprises the first nucleic acid molecule and the second nucleic acid molecule, or (c) a single nucleic acid molecule comprises the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule.
The present invention further contemplates molecular circuits comprising (a) a first nucleic acid molecule that comprises a gene encoding a first transcription factor and a first promoter activatable by stress, wherein the first promoter and the first transcription factor gene are operably linked, (b) a second nucleic acid comprising a gene encoding a second transcription factor and a second promoter activatable by the first transcription factor and the second transcription factor, wherein the second promoter and the second transcription factor gene are operably linked, and (c) a third nucleic acid molecule that comprises a gene of interest and a third promoter activatable by the second transcription factor, wherein the third promoter and the gene of interest are operably linked. These molecular circuits may comprise (a) three separate nucleic acid molecules, (b) the third nucleic acid molecule and a single nucleic acid molecule that comprises the first nucleic acid molecule and the second nucleic acid molecule, or (c) a single nucleic acid molecule comprises the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule.
The present invention also contemplates molecular circuits comprising (a) a first nucleic acid molecule that comprises a gene encoding a transcription factor and a first promoter activatable by stress, wherein the first promoter and the transcription factor gene are operably linked, and (b) a second nucleic acid molecule that comprises a gene of interest, the transcription factor gene, and a second promoter activatable by the transcription factor, wherein the second promoter is operably linked with the gene of interest and the transcription factor gene. These molecular circuits may comprise two nucleic molecules, or the molecular circuits may be contained within a single nucleic acid molecule that contains the first and second nucleic acid molecules.
In molecular circuits of the present invention, the transcription factor can be, for example, a mutated heat shock transcription factor (HSF) or a chimeric transcription factor. A suitable mutated HSF can be derived from a vertebrate HSF or from an insect HSF. For example, a suitable vertebrate HSF can be a mammalian HSF or an avian HSF.
The molecular circuits described herein can be contained within a single expression vector. Alternatively, molecular circuit nucleic acid molecules can be contained within a set of expression vectors, wherein each expression vector contains one or two molecular circuit nucleic acid molecules.
The present invention also contemplates recombinant host cells that comprise a molecular circuit. The molecular circuit may have the form of a single expression vector or an expression vector set, as described above. Suitable eukaryotic host cells include insect cells, avian cells, yeast cells, and mammalian cells.
The present invention further contemplates methods of producing a protein of interest, comprising the steps of: (a) culturing such recombinant host cells, (b) stimulating the first promoter by exposing the cultured recombinant cells to stress, and (c) isolating the protein of interest from the cultured recombinant host cells, wherein the protein of interest is expressed by the gene of interest. Step (b) can be achieved, for example, by heating the recombinant host cells.
The present invention also contemplates viruses that comprise an expression vector described above. Suitable viruses include adeno-associated viruses, adenoviruses, Herpes simplex viruses, alphaviruses, and pox viruses.
The present invention also includes pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and either an expression vector or an expression vector set, as described above. Alternatively, a pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier and a virus, as described above.
The present invention further contemplates methods of treating a subject with a protein of interest, comprising the steps of: (a) administering to a subject a pharmaceutical composition described above, and (b) applying heat to the area of the subject in need of the protein of interest, wherein the heat treatment results in the stimulation of the expression of the gene of interest.
The present invention also includes methods of stimulating the expression of a gene of interest in a recombinant cell, comprising the steps of: (a) producing a recombinant host cell by introducing into a host cell either the expression vector, or an expression vector set, as described above, and (b) exposing the recombinant host cell to a condition of stress, wherein the stress exposure stimulates the first promoter to increase expression of the gene operably linked to the first promoter, which in turn, results in the stimulation of expression of the gene of interest.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are identified below and are incorporated by reference in their entirety.